Project Summary Vascular calcification is associated with coronary artery disease and poor prognosis. This prompted us to examine whether calcification can be a pathogenic factor in atherosclerosis. We developed a mouse model, in which overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells prompted the development of complex calcified arterial lesions that originated as macro-calcific nodules in the intima. We then tested the idea that pathologic micro-calcification can precede atherosclerosis by challenging endothelial TNAP mice with high cholesterol diet. We documented that animals with pre-existing intimal calcification developed accelerated coronary artery disease, whereas equally hyperlipidemic mice without TNAP overexpression displayed a typical course of murine atherosclerosis, in which coronary arteries were unaffected. Pharmacological inhibition of TNAP under these conditions by a specific chemical inhibitor SBI-425 reduced coronary atherosclerosis, normalized heart fraction, and improved survival. Analyzing human myocardial samples, we detected TNAP activity in the endothelium of small coronary arteries and arterioles. Our significant findings regarding TNAP, calcification and atherogenesis merit further investigation of the role of this enzyme in vascular disease. Our overall hypothesis is that under conditions that promote pathologic vascular calcification (e.g. chronic kidney disease, diabetes, and advanced age), TNAP activity might induce micro-calcification in the intima that can accelerate lipid retention and inflammation and that inhibition of TNAP activity could be a valuable therapeutic goal for the reduction of atherosclerosis in patients predisposed to vascular calcification. Here we propose to determine the prevalence of intimal micro-calcification in a large sample of individuals over the age of 65; using 250 human cadavers specimens (Aim 1.1). Going back to the animal study and using computational flow dynamics (CFD) simulations, we will compute how micro-scale endothelial roughness (experimentally determined by submicron-resolution optical scanning) affects shear stress distribution. These experiments should extend our understanding of the relationship between vascular calcification and atherogenesis associated with low shear fields ? a fundamental pathophysiological process (Aim 1.2). A potential interaction between adenosine (another product of TNAP) and alpha adrenergic signaling in the regulation of vascular tone will also be examined to explain increased sensitivity of TNAP-positive arteries to sympathomimetic stimulation and impaired relaxation (Aim 2.3). In Aim 2, we will interrogate TNAP axis in a chronic kidney disease model both genetically, by targeted deletion of TNAP in endothelial cells (Aim 2.1), and pharmacologically, by SBI-425 (Aim 2.2). Upon completion of this work we will establish that TNAP- induced vascular calcification can mechanistically precede atherosclerosis. We will also validate TNAP inhibition as a viable pharmacological approach that could potentially be used to treat many human conditions associated with both increased calcification and atherosclerosis.